Recessive mutations in the Anoctamin-5 gene (ANO5, TMEM16E) cause Limb-Girdle Muscular Dystrophy 2L (LGMD2L), Miyoshi Muscular Dystrophy 3 (MMD3), and other generalized myopathies. ANO5 is a member of a 10-gene superfamily, the founding members of which (ANO1 and ANO2) are plasma membrane Ca2+-activated Cl- channels. Because ANO5 is 38% identical (54% similar) to ANO1, it is widely assumed that ANO5 is a Cl- channel and that ANO5 myopathies are explained by defects in ion transport. Recently, however, it has become apparent that some ANOs, notably ANO6 - which is 75% similar to ANO5, have an additional function: they stimulate phospholipid scrambling (PLS). PLS is the physiological loss of phospholipid asymmetry in the plasma membrane, typified by the translocation of phosphatidylserine (PtdSer) from its location in the cytoplasmic leaflet of the plasma membrane to the extracellular leaflet. The arrangement of PtdSer in the membrane is important for two reasons: PtdSer is known to serve as a platform for the assembly of membrane-associated protein complexes and is an important regulator of membrane fusion during endo- and exo- cytosis. This application tests the hypothesis that ANO5 is a phospholipid scramblase and an ion channel and then uses this information to explore the mechanisms of ANO5-associated skeletal muscle pathology. ANO5-myopathies, and related myopathies like ones caused by mutations in dysferlin, are explained by defects in mechanisms that repair membrane injury produced normally by exercise. Such injury is healed by two processes: (1) resealing of small lesions by assembly of new plasma membrane to fill the holes and (2) fusion of muscle progenitor stem cells (satellite cells) to regenerate new muscle fibers at sites of more severe damage. We propose that reorganization of membrane lipids mediated by ANO5 plays a fundamental role in these processes. There are three specific aims. (1) We will determine if ANO5 is a phospholipid scramblase, a regulator of a scramblase, and/or an ion channel. We will evaluate ion channel function by patch clamp and PLS by imaging fluorescent phospholipid probes in both HEK cells overexpressing ANO5 and in muscle cells endogenously expressing ANO5. (2) We will then investigate the cellular mechanisms of ANO5-mediated PLS in cultured myotubes and test whether ion transport plays a role. (3) We will elucidate the role of ANO5 in membrane repair using myotubes expressing wild type, disrupted, or mutant ANO5. Further, we will evaluate the function of pathogenic ANO5 variants to determine the functional consequences of human variations in ANO5 that are linked to myopathy. The effects of disease-associated ANO5 sequence variants on ion channel function, PLS, membrane repair, and myoblast fusion will be characterized in myotubes transfected with these variants. This study has the potential to open a completely novel line of investigation that may lead to new therapies for muscular dystrophies, especially those caused by ANO5 dysfunction, but potentially also other types of muscular dystrophies caused by muscle membrane fragility or defective repair.